Polymers

ABSTRACT

A polymer having a plurality of 5-6 membered unsaturated rings which may be the same or different, linked together by way of linker chains to form a backbone, at least some of said 5-6 membered unsaturated rings carrying a ligand. Polymers of this nature can act as DNA mimics and as supports for use in oligonucleotide production.

The present invention relates to polymers, in particular polymers which are useful as nucleic acid mimics or for use in the solid phase synthesis of oligomers.

DNA such as found in nature, if frequently used as a tool or probe in the detection of analytes such as biological substances, in particular in diagnosis and detection of microbiological organisms such as pathogenic bacteria.

Recently a nucleic acid mimic known which interacts with DNA and is known as peptide nucleic acid (PNA) has been developed. It is known that peptide nucleic acids will strand invade DNA at purine rich sites to form triplex structures (P. E. Nielson et al., Science, 1991, 254, p 1497-1506, Turney D. Y. et al,. Proc. Natl. Acad. Sci. USA, 1993, 90, 1667-1670).

PNA has a polymer or pseudopeptide backbone synthesized to mimic a strand of DNA in its configuration by having six bonds between nucleotide units and three bonds between the backbone and each nucleotide base. The advantages of using DNA mimics in detection methods are generally that these have increased detection limits with respect to naturally occurring DNA. The hybridization kinetics may be faster and the structures may be more robust or stable than DNA.

There are many applications of nucleic acid mimics in biotechnological research and in therapeutic applications, some of which are summarised for example in EP-A-0839830, together with an acyclic nucleotide mimic structure. Further polymers, useful for solid phase synthesis of oligomers are described in WO 92/07882.

The applicants have found that 5-6 membered unsaturated rings make good backbone elements for nucleic acid mimics and other polymers.

The present invention provides a polymer having a plurality of 5-6 membered unsaturated rings which may be the same or different, linked together by way of linker chains to form a backbone, at least some of said 5-6 membered unsaturated rings carrying a ligand.

In particular, the present invention provides a compound of general formula (I)

where n is an integer in excess of 1, each A is the same or different and is a 5 or 6 membered unsaturated ring, each R¹ is the same or different to any other such group and is selected from hydrogen or a ligand group, each R² is the same or different and is an optionally substituted linker group, R³ is hydrogen or a blocking group, R⁴ is at least a part of a linker group, p is 0 or 1, and R⁵ is a leaving group or a blocked derivative thereof.

Suitable 5-6 membered unsaturated rings A are C₅₋₆cycloalkenyl, C₅₋₆cycloalkynyl, phenyl or unsaturated heterocyclic rings. Heterocyclic rings may contain one or more heteroatoms. Suitable heteroatoms are oxygen, nitrogen or sulphur. Thus examples of suitable heterocyclic rings include triazine, pyridine, pyrimidine, pyrazine, imidazole, imidazoline, pyrazole, pyrrole, furan, thiazine and thiophene.

Preferably the 5-6 membered unsaturated rings are generally planar under normal conditions, and so preferred rings are aromatic rings such as phenyl or heteroaromatic rings such as triazine, pyridine, pyrimidine, pyrazine, imidazole, pyrazole, pyrrole, furan, thiazine and thiophene,

Most preferably, the unsaturated ring A is a 6-membered aromatic group of sub-formula (i)

where X₁, X₂ and X₃ are independently selected from C or N.

In particular, groups or sub-formula (i) are nitrogen-containing rings such as triazine, pyridine and pyrimidine, and of these, triazine is a most preferred group.

Suitable linker groups R² will vary depending upon the precise nature of the polymer required and the purpose to which it is to be put. Generally however, R² will comprise an optionally substituted chain of from 2-8 atoms or an optionally substituted ring of 3-8 atoms, where the atoms are selected from carbon atoms or heteroatoms such as nitrogen, oxygen and sulphur.

Where R² is a ring, it may be aromatic or non-aromatic. A particular example of an aromatic ring linker is benzyl and a particular example of a non-aromatic ring linker R²is cyclohexyl.

In particular the linker R² comprises an optionally substituted chain of 2-8 atoms and preferably 3-8 atoms.

Where linker groups R² include carbon or nitrogen atoms, these will be substituted with further groups to satisfy valency constraints and these groups may be hydrogen but may alternatively be an optional substituent.

Suitable optional substituents for linker groups R² include alkyl (such as C₁₋₄alkyl), hydroxy, hydroxyalkyl (such as hydroxyC₁₋₄alkyl and in particular hydroxymethyl) alkoxy (such as C₁₋₄alkoxy), alkanoyl (such as C₁₋₄alkanoyl), carboxy or salts or esters thereof (such as alkyl esters), carboxyalkyl (such as carboxyC₁₋₄alkyl and in particular CH₂COOH) or salts or esters thereof, (such as alkyl esters), nitro, nitroalkyl, such as C₁₋₄nitroalkyl in particular —CH₂NO₂) cyano, cyanoalkyl such as C₁₋₄cyanoalkyl in particular —CH₂CN), amino, mono- or di-alkyl amino, aminoalkyl (such as aminoC₁₋₄alkyl and in particular —CH₂NH₂) phosphate, alkyl phosphate (such as C₁₋₄alkyl phosphate and in particular CH₂PO₃), sulphate, alkyl sulphate (such as C₁₋₄alkyl sulphate and in particular CH₂SO₃), mercapto, alkylthio (such as C₁₋₄alkylthio), alkylsulphonyl, or a reporter molecule or tag.

The substituents on the linker groups may affect the lipophilicity or hydrophilicity of the desired polymer. For example, where lipophilic polymers are required, substituents may be omitted or may be selected from alkyl groups such as C₁₋₄ alkyl and in particular methyl. Where hydrophilic polymers are required, charged or electron-rich substituents such as hydroxy, hydroxyalkyl, carboxy or salts or esters thereof (such as alkyl esters), carboxyalkyl, nitro, cyano, phosphate, alkylphosphate, sulphate or alkylsulphate may suitably be used.

Where optional substituents for the linker groups R² include reporter molecules or tags, these may comprise fluorescent labels. Typical nucleic acid labels include fluorescein derivatives (such as 5-carboxyfluorescein, fluorescein isothiocyanates (FITCs), fluorescein dichlorotriazines (DTAIs)), rhodamine derivatives (such as 5-carboxytetramethylrhodamine, rhodamine 6G, Rhodamine Green™, Rhodamine Red™, Lissamine™, rhodamine B), Texas Red™, BODIPY™ and Cascade Blue™. Other stains which may be used for detection of nucleic acids include ethidium bromide, acridine homodimer, acridine orange, ethidium-acridine heterodimer, SYBR™ Green, SYBR™ Gold and PicoGreen™

Suitably the linking groups R² include both carbon and heteroatoms, in particular oxygen or nitrogen atoms.

Particularly preferred linking groups R² are groups of sub-formula (ii) or (iii) —Y₁—(CR⁶R⁷)_(m)—Y₂—  (ii) —(CR⁸R⁹)_(s)—Y³—(CR¹⁰R¹¹)_(t)—  (iii)

where m, s and t are independently selected from integers of from 1-6 and preferably 2-4, and Y₁ and Y₂ are independently selected from a bond, O, S, NR¹², —C(O)O—, —OC(O)—, —NR¹²C(O)—, —C(O)NR¹²—, or —NR¹²C(O)NR¹³ where R¹² and R¹³ are independently selected from hydrogen or C₁₋₄alkyl such as methyl, and Y³ is a group which is formed by joining two moieties together, such as esters and amides of formulae —C(O)O—, —OC(O)—, —NR¹²C(O)—, —C(O)NR¹²—, or —NR¹²C(O)NR¹³ where R¹² and R¹³ are as defined above; and each R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ is independently selected from hydrogen, alkyl (such as C₁₋₄alkyl), hydroxy, hydroxyalkyl (such as hydroxyC₁₋₄alkyl and in particular hydroxymethyl) alkoxy (such as C₁₋₄alkoxy), alkanoyl (such as C₁₋₄alkanoyl), carboxy or salts or esters thereof (such as alkyl esters), carboxyalkyl (such as carboxyC₁₋₄alkyl and in particular CH₂COOH) or salts or esters thereof, (such as alkyl esters), nitro, nitroalkyl, such as C₁₋₄nitroalkyl in particular —CH₂NO₂) cyano, cyanoalkyl such as C₁₋₄cyanoalkyl in particular —CH₂CN), amino, mono- or di-alkyl amino, aminoalkyl (such as aminoC₁₋₄alkyl and in particular —CH₂NH₂) phosphate, alkyl phosphate (such as C₁₋₄alkyl phosphate and in particular CH₂OPO₃), sulphate, alkyl sulphate (such as C₁₋₄alkyl sulphate and in particular CH₂SO₃), mercapto, alkylthio (such as C₁₋₄alkylthio), alkylsulphonyl, or a reporter molecule or tag as described above.

Suitably at least some groups R⁶, R⁷, R⁶, R⁹, R¹⁰ and R¹¹ are hydrogen. Most preferably, a maximum of one group R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ is other than hydrogen.

Preferably, R¹² and R¹³ are hydrogen.

As used herein, the term “alkyl” includes both straight and branched chain groups, which, unless otherwise stated may contain for example from 1-10 and suitably from 1-6 carbon atoms. Other terms, such as alkoxy and alkanoyl used herein will include alkyl groups as discussed here, combined with the conventional functionality.

Particular examples of linking groups of sub-formula (ii) are groups —NHCH₂CH₂NH—, —OCH₂CH₂—, —NHC(O)CH₂—, —NHC(O)NHCH₂—, —NHC(OH)CH₂NH—, —NHCH(CH₂COOH)CH₂NH—, —NHCH(CH₂OH)CH₂NH₂—,  OCH(CH₂OH)CH₂O—, —NHCH(CH₂OPO₃ ⁻²)CH₂NH₂—, or —NHCH(CH₂NH₂)CH₂NH—. A particularly preferred example of a linking group R² is a group —NHCH₂CH₂NH—. Such linking groups will be useful in particular where lipophilic polymers are required.

Examples of suitable ligands for R¹ will depend upon the nature of the polymer and the purpose to which it is going to be put. Ligands R¹ may be selected for example from nucleobases, and in particular naturally occurring nucleobases, nucleobase binding groups or DNA intercalators. These may be bound directly to the ring A, or a spacer group such as a C₁₋₄ and preferably a C₁₋₂ alkylene chain may be interposed between the nucleobases, nucleobase binding group or DNA intercalator and the ring A.

Such ligands will allow the compound of formula (I) to form interactions, such as hybridisation interactions, with DNA or RNA molecules.

For use as DNA mimics however, the ligands R¹ will generally comprise naturally occurring nucleobases. However, in some instances, where additional spacing between ligands is required, at least some of the groups R¹ may be hydrogen. Preferably however, most if not all groups R¹ will be other than hydrogen.

Generally speaking, the nature of R³, R⁴ where present, and R⁵ will depend upon the production process used in the manufacture of the polymer as outlined below, as well as the nature of the linking group R². Suitable blocking groups R³ are those used to protect groups Y² in sub-formula (ii) above during the polymer production reaction. Thus for example, R³ may be an alkylcarbonyl or arylcarbonyl such as benzyl carbonyl group when Y² is oxygen, or an alkoxy carbonyl ester such as t-butoxy carbonyl ester when Y² is NH—. Commonly however, R³ will be hydrogen.

Similarly, R⁵ may be a leaving group such as halogen such as fluoro, chloro, bromo or iodo, as well as mesylate or tosylate, and in particular chloro. Such leaving groups may be required in the production process to allow the polymer chain to build up in that direction if required. However, the terminal leaving groups may be blocked if desired or necessary to prevent chain extension in that direction, using conventional chemistry as described hereinafter.

In the compounds of formula (I), the integer n may be anything depending upon the nature of the product being produced. Generally speaking however, n will be in the range of from 2-200 and preferably from about 6-100, more preferably 10-60.

In a particular embodiment, the compounds of the invention are compounds of formula (II)

where R², R³, R⁵ and n are as defined in relation to formula (I), each R¹ is the same or different and is as defined in relation to formula (I), and X₁, X₂ and X₃ are as defined in relation to sub-formula (ii).

Particularly suitable compounds of formula (II) can be partially represented as follows:

where B₁, B₂ and B3 represent nucleobases

a, b and c are independently selected from 0 or an integer of from 1-6, provided that a+b+c is equivalent to m as defined in relation to sub-formula (iii);

each R²⁰ is an optional substituent for a linker group as defined above;

R⁵ is as defined in relation to formula (I),

Y₁ and Y₂ are as defined in relation to sub-formula (iii) and the polymer extends further in a similar fashion in the direction of the dotted line.

Preferably, in the above formula, b is 0, 1 or 2 and preferably 0 or 1.

Suitably, a and c are independently selected from 0, 1 or 2.

Preferably, where b is 1, one of a or c is 0 and the other is 1.

Where R⁵ is a leaving group, further chain growth at that end of the polymer is facilitated.

Polymeric compounds of formula (I) are suitably prepared by processes which are conventional in the literature. In particular, they may be prepared by consecutively adding appropriately substituted monomers to a chain. Thus, compounds of formula (I) are suitably prepared by a reiterative process comprising the following steps:

(a) reacting a compound of formula (III)

in which A, R⁴, R⁵ and p are as defined in relation to formula (I),

R^(1′) is a group R¹ as defined in relation to formula (I) or a protected derivative thereof;

R^(2′) is a group R² as defined in relation to formula (I) or a protected form thereof, where p is 0, or R^(2′) is a precursor group, such that R² or a protected form thereof is formed by —R^(2′)—R⁴— where p is 1, and

R¹⁴ is a protecting group,

with a compound of formula (IV);

where A, R² R⁴ and p are as defined in relation to claim 1, q is 0 or 1, R^(1′) and R^(2′) are as defined in relation to formula (III), and all of these may be the same or different to the corresponding moieties in the compound of formula (III), where R¹⁵ is a protecting group and q is 0 or 1, then

(b) removing a protecting group R¹⁴ and reacting the product with a further compound of formula (III); or

(c) where q is 1, removing a protecting group R¹⁵ and reacting the product with a further compound of formula (III); and

(d) repeating step (b) and/or (c) until the desired polymer is obtained;

(e) removing any protecting groups on R^(1′) and/or R^(2′); and thereafter if desired or necessary, removing any remaining protecting groups R¹⁴ or R¹⁵ and/or introducing a blocking group R³.

In this process therefore, the total number of stages in steps (a), (b) and/or (c) will total n-1 where n is as defined in relation to formula (I).

In each step, the compound of formula (III) employed may be the same or different. Where these are different, the precise structure of the target polymer of formula (I) can be selected.

For example, by varying the nature of the linker groups included in the polymer chains, points of particular weakness can be included, which may then be subject to cleavage thereafter if required.

The coupling reactions of the above process are suitably effected under conventional conditions. For example, the coupling is suitably effected in a solvent, and in particular an organic solvent such as tetrahydrofuran (THF) at moderate temperatures which may be in the range of from 0 to 80° C., preferably from 30 to 50° C. The reaction may be effected in the presence of a base which is suitably a weak base such as triethylamine.

Where p is 1, the coupling results in the formation of the linker group R². For example, it may be particular suitable to form a bond such as an amide bond in the middle of the linker R², which may therefore be of sub-formula (iii) as defined above. Thus in this case, a particularly suitable group R^(2′) in formula (IV) would comprise a group of sub-formula (iv) —(CR⁸R⁹ )_(s)—NH—  (iv)

where R⁸, R⁹ and s are as defined in relation to sub-formula (iii), and in the compound of formula (III), R⁵ is hydroxy, and R⁴ is a group of sub-formula (v) —C(O)—(CR¹⁰R¹¹)_(t)—  (v)

where R¹⁰, R¹¹ and t are as defined in relation to sub-formula (iii).

The precise nature of the protecting groups R¹⁴ and R¹⁵ will vary depending upon the moiety they are attached to (i.e R^(2′) or R² respectively), and suitable examples would be apparent to a skilled chemist. Preferably R¹⁴ is different to R¹⁵ to facilitate removal of one of these groups at a time. Suitable protecting groups R¹⁴ and R¹⁵, (for example where R^(2′) and/or R² include amino groups) include benzyl, alkoxycarbonyl esters such as t-butoxycarbonyl ester (Boc),9-fluorenylmethoxycarbonyl ester (FMOC), or amides such as N-formyl, or imides such as phthalimide. Where R^(2′) and R² contain oxygen atoms such as ethers, R¹⁴ and/or R¹⁵ are suitably esters such as acetates in particular OC(O)CH₃ or trifluoroacetate, carbamates such as alkyl carbamates in particular —OC(O)OCH₃, or ethers such as tertiary butylether, benzylether, or trimethylsilylether.

Similar considerations apply with respect to the presence or otherwise of protecting groups on the group R^(1′) and/or R^(2′). For example, where R¹ is a naturally occurring nucleobase, it may be preferable to protect one or more of the amino groups or ring nitrogen atoms in order to ensure that no accidental “cross-linking” of the polymer may occur during formation. Suitable protecting groups include alkanoyl or aroyl groups such as benzoyl. This are suitably introduced onto the base as a preliminary step using conventional methods for example as illustrated hereinafter.

Where R^(2′) contains reactive substituents such as hydroxy, amino or carboxy groups, these may be protected during the preparation using methods similar to those outlined in relation to R¹⁴ and R¹⁵.

Removal of protecting groups R¹⁴ and R¹⁵ and any protecting groups present on R^(1′) or R^(2′) may be effected under conventional conditions also as illustrated in the examples given hereinafter.

Compounds of formula (III) and (IV) are suitably prepared by introducing the appropriate substituents, in any order, onto compound of formula (V)

where A is as defined in relation to formula (I) and R¹⁷, R¹⁸ and R¹⁹ are the same or different and are each leaving groups. Examples of suitable methods are given hereinafter. The order in which the substituents are introduced into the compound of formula (V) will vary depending upon the nature of the particular substituents involved. Preferably, R¹⁷, R¹⁸ and R¹⁹ are different to each other as this allows precise targeting of substituents around the ring. However, the applicants have found that two or more of R¹⁷, R¹⁸ and R¹⁹ may be the same and that careful control of reaction conditions provides good yields of the desired substituted product.

Suitable leaving groups R¹⁷, R¹⁸ and R¹⁹ include leaving groups as listed above in relation to R⁵, and in particular are halo groups such as chloro.

For example, compounds of formula (III), where p is 0 and R⁵ is the same leaving group as R¹⁹ are suitably prepared by reacting a compound of formula (VI)

R² is as defined in relation to formula (I), R¹⁴ is as defined in relation to formula (III), and R¹⁷ and R¹⁹ are as defined in relation to formula (V), with a compound of formula (VII) HR^(1′)  (VII)

where R^(1′) is as defined in relation to formula (III). The reaction is suitably effected in a solvent such as an acetone/water solvent, in the presence of a base such as sodium carbonate. Moderate temperatures, for example from 0° to 50° C., and conveniently about room temperature, are employed, in order to avoid over production of an unwanted trisubstituted product, which may be a particular risk, where R¹⁷ and R¹⁹ are the same leaving groups.

Compounds of formula (VI) may be prepared by reacting a compound of formula (V) as defined above, with a compound of formula (VIII) H—R²—R¹⁴   (VIII)

where R² is as defined in relation to formula (I), and R¹⁴ is as defined in relation to formula (III). The reaction is suitably effected in an organic solvent such an acetone/water solvent, in the presence of a base such as sodium hydrogen carbonate. In this case, low temperatures, for example from −20° to 10° C., and conveniently about 0° C., are employed, in order to avoid over production of an unwanted di- or tri-substituted products

In a particular embodiment, the invention provides a method of preparing a compound of formula (X)

where R² is as defined in relation to formula (I) and R¹⁴ is as defined in relation to formula (III), by reacting a cyanuric chloride with a compound of formula (XI) H—R²—R¹⁴   (XI)

were R² and R¹⁴ are as defined above, at a temperature below 10° C., and preferably at about 0° C.

Compounds of formula (III) may be converted to compounds of formula (IV), by reaction with a compound of formula (IX) R¹⁵

R²

_(q)H   (IX)

where R² is as defined in relation to formula (I), and R¹⁵ and q are as defined in relation to formula (IV). The reaction is suitably effected in an organic solvent such as tetrahydrofuran (THF), at elevated temperatures, for example at the reflux temperature of the solvent.

Intermediates of formula (III), (IV) and (VI) may be novel compounds and as such, form a further aspect of the invention.

Compounds of formula (V), (VIII) and (IX) are either known compounds or they can be prepared from known compounds by conventional methods.

Processes for the production of compounds of formula (I) may be subject to automation if required.

Compounds of formula (I) are particularly good DNA mimics in spite of the fact that both the ring structure A and the linker group differ significantly from those found in DNA as illustrated below, where “TNA” is a preferred example of a compound of the invention, B represents a nucleobase, and Y₁ and Y₂ are as defined in relation to sub-formula (iii) above.

This means that they find wide applications, in biotechnology including oligomer production techniques, and also in therapeutic applications.

The invention will now be particularly described by way of example.

EXAMPLE 1

Step 1

Protection of Ethylenediamine

Ethylene diamine (10.45 g) in dichloromethane (60 ml) was added to a solution of t-butoxy carbonyl ester (Boc) anhydride (4.90 g) in dichloromethane (60 ml) over a 2 hour period. The reaction was stirred for 22 hours at room temperature before the solvent was evaporated. Water (100 ml) was added to the residue and the insoluble product was removed by filtration. The aqueous solution was extracted with dichloromethane (3×100 ml) and the combined organic layers were evaporated to afford the desired monoprotected product as a colorless oil (23.01 g, 83%).

Step 2 Attachment of the Mon-Boc Protected Ethylenediamine to Cyanuric Chloride

The N-Boc-ethylenediamine from step 1 (500 mg) was added to a slurry of cyanuric chloride (75 mg) in acetone (12 ml) which was then poured into ice water held at 0° C. with stirrinc. Sodium bicarbonate (262 mg) was added and after stirring for 2 hours at 0° C., a white solid was filtered off, washed with water and dried in vacuo over P₂O₅. Care was taken to ensure that the temperature of the reaction did not go above 0° C. to prevent formation of unwanted di- and tri-substituted products and hydrolysis of the chloro groups. The desired product was obtained as a white solid (763 mg, 93%) which was recrystallized from pyridine.

Step 3 Monoprotection of the Nucleobase, (Thymine) as the Benzoylamino Derivative

Thymine (2.62 g) was dissolved in acetonitrile (33 ml) and pyridine (13.5 ml) and benzoyl chloride (11.68 g) was added. The reaction mixture was stirred at room temperature for 24 hours. The resultant orange solution was evaporated to dryness and the residue extracted into a 1:1 dioxane/water mixture (62 ml). Potassium carbonate (4.86 g) was added and the suspension was stirred at room temperature overnight. Hydrochloric acid (1N) was added to the solution to reduce the pH to about 3, whereupon the precipitate was collected and recrystallised from ethanol. The desired 3-benzoylamino derivative was obtained as pale yellow needles (2.5 g).

Step 4 Attachment of the Nucleobase to the Monosubstituted Product

The 3-N-benzoyl thymine from step (3) (0.4 g), sodium carbonate (0.2 g) and the monosubstituted product of step 2 (0.5 g) in acetone (20 ml) and water (10 ml) were mixed together. The reaction mixture was then stirred at room temperature for 5 hours. Completion of the reaction was indicated by TLC. The resultant yellow precipitate was filtered off and dried to give the desired product as a pale yellow solid (0.41 g).

Step 5 Monoprotection of Ethylenediamine as the Benzylamino Derivative

A second monomer was prepared which introduced a third substituent onto the triazine molecule. Ethylene diamine (10 g) in dichloromethane (60 ml) was mixed with benzylchloride (21.01 g)in dichloromethane. The mixture was stirred at room temperature for some hours and the solution was concentrated under reduced pressure. The residue was dissolved in ethylactate (100 ml) and the resultant solution was washed with 1M potassium hydrogen sulphate (2×100 ml), 5% sodium hydrogen sulphate (100 ml), and brine (100 ml), and then dried over magnesium sulphate. The solvent was evaporated under reduced pressure and the desired product was obtained as a colourless oil (22.40 g, 92%).

Step 6 Trisubstitution of the Disubstituted Triazine Derivative

The disubstituted derivative produced in step 4 above (0.1 g) suspended in tetrahydrofuran (THF) (5 ml) was treated with the benzyl protected ethylenediamine compound (0.034 g) from step 5. The reaction mixture was heated under reflux overnight and the reaction was deemed to be complete when no further starting material was observed by TLC. The off-white solid that resulted was filtered and washed with more THF. After recrystallisation from pyridine, the desired product was obtained as an off-white solid (84 mg, 63%).

Step 7 Preparation of Dimer

The two monomers produced in steps 4 and 6 respectively were then be coupled in a stepwise fashion in solution as illustrated in the following reaction Scheme.

In particular, the trisubstituted product of step 6 (0.50 mg) was suspended in dichloromethane (300 ml) and trifluoroacetic acid (0.5 ml) added. The mixture was stirred for 1 hour and then evaporated. The residue was dissolved in THF (5 ml) with triethylamine (24.52 ml) and the disubstituted product of step 4 (34 mg) was added. The mixture was stirred for 2 hours at 40° C. and further triethylamine added until the mixture was basic. Volatile materials were then removed under reduced pressure. The residue was dissolved in ethylacetate, washed with potassium hydrogen sulphate (2×5 ml), water (5 ml), sodium bicarbonate (2×5 ml) and brine and dried over magnesium sulphate. Solvent was then removed under reduced pressure to yield the desired product as an off-white solid (70.91. 93%).

The process of step 7 could be repeated with the same or different monomers until an optimum chain is obtained. 

1-18. (canceled)
 19. A process for preparing a polymer having a plurality of 5-6 membered unsaturated rings which may be the same or different, linked together by way of linker chains to form a backbone, at least some of said 5-6 membered unsaturated rings carrying a ligand, which process comprises consecutively adding appropriately substituted monomers to a chain.
 20. A process according to claim 19 in which the polymer comprises a compound of general formula (I)

where n is an integer in excess of 1, each A is the same or different and is a 5 or 6 membered unsaturated ring, each R¹ is the same or different to any other such group and is selected from hydrogen or a ligand group, each R² is an optionally substituted linker group which may be the same or different to any other such group, R³ is hydrogen or a blocking group, R⁴ is at least a part of a linker group, p is 0 or 1, and R⁵ is a leaving group or a blocked derivative thereof.
 21. A process according to claim 20 which comprises the following steps: (a) reacting a compound of formula (III)

in which A is a 5 or 6 membered unsaturated ring, R⁴ is at least a part of a linker group, R⁵ is a leaving group or a blocked derivative thereof, p is 0 or 1, R^(1′) is selected from hydrogen or a ligand group or a protected derivative thereof; R^(2′) is an optionally substituted linker group or a protected form thereof where p is 0, or R^(2′) is a precursor group, such that an optionally substituted linker group or a protected form thereof is formed by —R^(2′)—R⁴— where p is 1, and R¹⁴ is a protecting group, with a compound of formula (IV); where A is a 5 or 6 membered unsaturated ring, R² is an optionally substituted linker group which may be the same or different to any other such group, R⁴ is at least a part of a linker group, p is 0 or 1, q is 0 or 1 and R^(1′) and R^(2′) are as defined above in relation to formula (III), and all of these may be the same or different to the corresponding moieties in the compound of formula (III), where R¹⁵ is a protecting group then (b) removing a protecting group R¹⁴ and reacting the product with a further compound of formula (III); or

(c) where q is 1, removing a protecting group R¹⁵ and reacting the product with a further compound of formula (III); and (d) repeating step (b) and/or (c) until the desired polymer is obtained; (e) removing any protecting groups on R^(1′) and/or R^(2′); and thereafter if desired or necessary, removing any remaining protecting groups R¹⁴ or R¹⁵ and/or introducing a blocking group R³.
 22. A process according to claim 19 which is carried out in an automated fashion.
 23. A process according to claim 20 which is carried out in an automated fashion.
 24. A process according to claim 20 wherein the rings A are aromatic rings.
 25. A process according to claim 20 wherein the rings A are of sub-formula (i)

where X₁, X₂ and X₃ are independently selected from C or N.
 26. A process according to claim 25 wherein at least some of the groups or sub-formula (i) are triazine groups.
 27. A process according to claim 20 wherein linker groups R² are independently selected from a chain of from 2-8 atoms or a ring of from 3-8 atoms, selected from carbon atoms or heteroatoms which may be optionally substituted.
 28. A process according to claim 27 wherein at least some linking groups R² are groups of sub-formula (ii) or (iii) —Y¹—(CR⁶R⁷)_(m)—Y²—  (ii) —(CR⁸R⁹)_(s)—Y³—(CR¹⁰R¹¹)_(t)—  (iii) where m, s and t are independently selected from integers of from 1-6, and Y¹ and Y² are independently selected from a bond, O, S, NR¹², —C(O)O—, —OC(O)—, —NR¹²C(O)—, —C(O)NR¹²—, or —NR¹²C(O)NR¹³ where R¹² and R¹³ are independently selected from hydrogen or C₁₋₄alkyl, and Y³ is a group which is formed by joining two moieties together, such as esters and amides of formulae —C(O)O—, —OC(O)—, —NR¹²C(O)—, —C(O)NR¹²—, or —NR¹²C(O)NR¹³ where R¹² and R¹³ are as defined above; and each R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ is independently selected from hydrogen, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkanoyl, carboxy or salts or esters thereof, carboxyalkyl or salts or esters thereof, nitro, nitroalkyl, cyano, cyanoalkyl, amino, mono- or di-alkyl amino, aminoalkyl phosphate, alkyl phosphate, sulphate, alkyl sulphate, mercapto, alkylthio, alkylsulphonyl, or a reporter molecule or tag.
 29. A process according to claim 28 wherein at least some linking groups R² are groups of formula —NHCH₂CH₂NH—.
 30. A process according to claim 19 wherein the ligand is selected from nucleobases, nucleobase binding groups or DNA intercalators, where the nucleobases, nucleobase binding groups or DNA intercalators may be bonded directly to the ring or may have a C₁₋₄alkylene chain interposed therebetween.
 31. A process according to claim 30 wherein at least some groups R¹ are naturally occurring nucleobases.
 32. A process according to claim 20, wherein the polymer is a compound of formula (II)

where R², R³, R⁵ and n are as defined in claim 2, each R¹ is the same or different and is as defined in claim 20, and X₁, X₂ and X₃ are independently selected from C or N.
 33. A compound of formula (III) as defined in claim
 21. 34. A compound of formula (IV) as defined in claim
 21. 35. A compound of formula (VI)

where R² and R¹⁴ are as defined in claim 20 and R¹⁷ and R¹⁹ are leaving groups.
 36. A method of preparing a compound of formula (X)

where R² is as defined in relation to formula (I) and R¹⁴ is as defined in relation to formula (III), by reacting a cyanuric chloride with a compound of formula (XI) H—R²—R¹⁴   (XI) where R² and R¹⁴ are as defined above, at a temperature below 10° C. 